Inhibition of vascular smooth muscle cell proliferation

ABSTRACT

Methods of treating a mammal having a condition characterized by vascular smooth muscle cell proliferation, where the mammal is treated with progesterone or an agonist thereof. Also disclosed are methods of screening compounds useful for inhibiting vascular smooth muscle cell proliferation.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] Under 35 USC §119(e)(1), this application claims the benefit ofprior U.S.. provisional application serial No. 60/044,651, filed Apr.18, 1997.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] The invention was funded in part by National Institutes of HealthGrant R01GM 53249. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] This invention relates to cardiovascular disease.

BACKGROUND OF THE INVENTION

[0004] Cardiovascular disease, the principal cause of death in thedeveloped world, affects men far more frequently than premenopausalwomen of the same age. Because the incidence of cardiovascular diseasein postmenopausal women gradually approaches that in age-matched men(McGill et al., Atheroscler. Rev. 4, 157-242 (1979)), estrogen orprogesterone appears to have a protective effect in premenopausal women.A large, 10-year prospective study supports such a cardioprotectiveeffect of estrogen by showing that postmenopausal estrogen-replacementtherapy reduces both the incidence of coronary heart disease andmortality from cardiovascular disease (Stampfer, M. J. et al., N. Engl.J. Med. 325, 756-762 (1991)).

[0005] As for the effects of progesterone on cardiovascular disease,however, there has been little information until the recent report byGrodstein et al. (N. Engl. J. Med. 335, 453-461 (1996)) showing that therisk of coronary heart disease is significantly lower in women who takeestrogen and progesterone together rather than estrogen alone. Estrogenadministration inhibits the development of experimentally inducedatherosclerosis in rodent models (Stamler et al., Circ. Res. 1, 94-98(1953); Weigensberg et al., Atherosclerosis 52, 253-265 (1984); andSullivan et al., J. Clin. Invest. 96, 2482-2488 (1995)). Also, incastrated baboons receiving estradiol and progesterone together, thereare fewer vascular lesions than in those receiving estradiol alone(Kushwaha et al., Arterioscler. Thromb. 11, 23-31 (1991)). However,there is little evidence of an independent effect of progesterone inanimal studies or in cell culture.

[0006] Current methods of treating severe cardiovascular conditions,such as coronary stenosis and occlusion caused by atherosclerosis,include invasive cardiovascular surgical procedures [e.g., percutaneoustranslumenal coronary angioplasty (“PTCA”) and aorta-coronary bypasssurgery (“ACBS”)]. However, the cellular proliferative response andassociated intimal hyperplasia that may follow the damage to bloodvessels caused by these procedures leads to complications which cannotbe effectively controlled by presently available drugs, and cantherefore be more detrimental than the original condition. Thedevelopment of these complications, termed restenosis (in the case ofPTCA) or stenosis (in the case of ACBS), has similarities to thedevelopment of atherosclerosis.

SUMMARY OF THE INVENTION

[0007] The invention is based on Applicants' discovery that (1)progesterone has a direct and specific inhibitory effect on arterialsmooth muscle cell proliferation; (2) this inhibition occurs at aprogesterone concentration that overlaps the physiological range ofprogesterone concentrations in the plasma of premenopausal women; (3)arterial smooth muscle cells contain abundant progesterone receptors(“PgR”) that mediate this effect; and (4) in the presence ofprogesterone, mRNA levels of cyclins A and E decline in rat arterialsmooth muscle cells (“RASMC”), suggesting that progesterone interruptsthe cell cycle at the G1/S transition. To Applicants' knowledge, this isthe first demonstration that progesterone exerts a direct inhibitoryeffect on vascular smooth muscle cell proliferation.

[0008] Applicants' discovery provides a biochemical explanation for therecent epidemiological observation that the relative risk of majorcoronary heart disease was 0.39 in postmenopausal women who tookestrogen with progesterone, as compared with a risk of 0.60 in those whotook estrogen alone (where the risk among women who took no hormones wasset at 1.0) (Grodstein et al, supra).

[0009] Accordingly, the present invention features a method oftreatment. In this method, one first identifies a mammal that issuspected of having, or at a risk of having, a condition characterizedby vascular smooth muscle cell (“VSMC”) proliferation and that is notbeing treated with estrogen or an estrogen agonist. Such a mammal can,for example, be a mouse, rat, dog, cat, cow, pig, horse, goat, sheep,rabbit, guinea pig, hamster or primate, such as a an adult human (e.g.,a man, a premenopausal woman, or a postmenopausal woman who is notpresently on an estrogen replacement therapy). A mammal is at a risk ofhaving the condition when it, for example, is at an age of beingsusceptible to the condition, or is genetically predisposed to or has afamily history of the condition, or is about to undergo or has recentlyundergone vascular surgery. A condition marked by VSMC proliferation istypically a cardiovascular or coronary heart disease such as transplantarteriosclerosis, atherosclerosis, angioplasty restenosis, or cardiacvein bypass stenosis.

[0010] After such a mammal is identified, one can then administer to themammal progesterone or a progesterone agonist in an amount effective todecrease VSMC proliferation in the mammal. A progesterone agonist is acompound that mimics, to various degrees, the effects of progesterone.This compound can be, for instance, an organic compound, a peptide, or anucleic acid.

[0011] An amount of progesterone or an agonist thereof is effective whenits administration results in inhibition of VSMC proliferation in ablood vessel in the mammal.

[0012] Also embraced by the invention are methods of identifyingcompounds potentially useful for inhibiting VSMC proliferation. In thesemethods, a test compound is contacted with a progesterone receptor(PgR). There are at least two isoforms of PgRs: PgR A and PgR B, themolecular weights of which are approximately 94 and 114 kD,respectively. The test compound's binding to the PgR is an indicationthat the compound is potentially useful for inhibiting vascular smoothmuscle cell proliferation. The PgR can be in a purified proteinpreparation, in a cellular extract, or inside cells [e.g., VSMCs orcells (e.g., mammalian, yeast, insect, or bacterial cells) that havebeen transfected with a DNA construct directing expression of the PgR].

[0013] Alternatively, the potential usefulness of a test compound isindicated by its ability to activate the PgR in a cell or in a cellextract. Activation of the PgR is detected, for example, by thephosphorylation status of the PgR, or by enhanced transcription of agene that is linked to a cis progesterone-responsive DNA element (e.g.,the hormone response element of the mouse mammary tumor virus). Thisgene can be an endogenous gene such as cyclin A or E, whosetranscription is downregulated by activated progesterone receptors (seeExample, infra) . Alternatively, the gene is an exogenously introducedreporter gene whose expression can be readily determined by abiochemical assay (e.g., a gene that encodes a firefly luciferase,β-galactosidase, or alkaline phosphatase) or by drug resistance of thecell harboring the gene (e.g., a gene that encodes chloramphenicolacetyltransferase, neomycin phospho-transferase, or guanine xanthinephosphoribosyltransferase).

[0014] The invention also provides methods of determining whethercompounds known or suspected to bind a PgR, such as known progesteroneagonists (e.g., norethisterone, or norgestrel), are potentially usefulfor inhibiting VSMC proliferation. In these methods, mammalian cells(e.g., mammalian VSMCs) containing a PgR are incubated with thecompounds and relative proliferation of the cells is determined (e.g.,by the amount of radioactive nucleotides incorporated into replicatinggenomic DNA, or by the number of cells generated by cell division). Thepotential usefulness of PgR-binding compounds can also be determined bymeasuring their ability to decrease cyclins A and/or E activity orexpression level (e.g., amounts of proteins or mRNA transcripts) inmammalian cells (e.g., mammalian VSMCs) containing a PgR.

[0015] Cells used to practice the invention can be freshly isolated fromtissues or cultured from isolated tissue cells. The cells can also bethose that have been immortalized in vivo (i.e., cancer cells) or invitro. A PgR used in the invention can be either naturally occurring(i.e., encoded by an endogenous gene) or recombinant, and need not befull length, so long as it contains the domains essential for itsfunctions.

[0016] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intended to be limiting.

[0017] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIGS. 1a-f are a set of photographs showing localization of PgRsin human arterial smooth muscle cells (“HASMC”s) and rat arterial smoothmuscle cells (“RASMC”s) (original magnification: 400×). FIGS. 1a-c showsequential sections of arterial tissue from a premenopausal woman. InFIG. 1a, a number of nuclei were positively stained for the PgR (blackspots, filled arrow), but some (open arrow) showed no PgRimmunoreactivity. In FIG. 1b, the absence of black spots afterpreabsorption with the PgR antigen demonstrates the specificity of theanti-PgR antibody staining. FIG. 1c shows that, in tissue double-stainedfor the PgR and α-actin (pink), PgRs localized to HASMCs. In FIG. 1d, anumber of nuclei were also positively stained for the PgR in a ratarterial tissue section. In FIGS. 1e and f, PgRs and α-actin colocalizedin cultured HASMCs and RASMCs, respectively.

[0019]FIGS. 2a-2 d are graphs showing effect of progesterone on [³H]thymidine incorporation in RASMCs or HASMCs. Thymidine incorporation isexpressed as a percentage of the value for the control (PBS-treated)group (100%). FIG. 2a, Dose-dependent inhibition of [³H] thymidineincorporation in RASMCs by progesterone. FIG. 2b, Progesterone-inducedinhibition of [³H] thymidine incorporation blocked by the PgR antagonistRU486. FIG. 2c, Effect of FCS concentration on inhibition of [³H]thymidine incorporation induced by progesterone at 500 nM. FIG. 2d,Dose-dependent inhibition of [³H] thymidine incorporation in HASMCs byprogesterone. Results from representative experiments are shown. Foursamples were analyzed in each experiment, and values represent the (mean±SEM). Comparisons were subjected to ANOVA followed by Fisher's leastsignificant difference test. Significance was accepted at P<0.05. *,Progesterone-treated group different from control group (100%). §,Progesterone-treated group different from RU486-treated group andRU486+progesterone-treated group.

[0020]FIG. 3a is a graph showing the effect of progesterone on RASMCproliferation. Progesterone was added at 500 nM to Dulbecco's modifiedEagle's medium (“DMEM”) supplemented with 1% (filled squares) or 2%(filled circles) charcoal/dextran-treated fetal calf serum (“C/D FCS”).Vehicle was added also at 500 mM to DMEM supplemented with 1% (opensquares) or 2% (open circles) C/D FCS.

[0021]FIG. 3b is a graph showing the dose-dependent inhibition of RASMCgrowth by progesterone. Three samples were analyzed in each experiment.Values represent the mean ±SEM. Comparisons were subjected to ANOVAfollowed by Fisher's least significant difference test. Significance wasaccepted at P<0.05. *, Progesterone-treated group different fromrespective vehicle control group (100%).

[0022]FIG. 4 is a set of autoradiographs showing Northern blot analysisof cyclin mRNAs expressed in the presence of the indicated doses ofprogesterone.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The invention provides a method of treating a mammal withprogesterone or a progesterone agonist. Any known progesterone agonistis a candidate for use in this treatment method; the efficacy of theagonist compound can be tested in vitro using the methods of theinvention and/or in vivo using animal models.

[0024] Progesterone or its agonist can be administered alone or in apharmaceutically acceptable carrier (e.g., physiological saline), whichis selected on the basis of the mode and route of administration, andstandard pharmaceutical practice. Suitable pharmaceutical carriers, aswell as pharmaceutical necessities for use in pharmaceuticalformulations, are described in Remington's Pharmaceutical Sciences, astandard reference text in this field, and in the USP/NF. Progesteroneor its agonists can be administered in dosages determined to beappropriate by one skilled in the art. It is expected that the dosageswill vary, depending upon the pharmacokinetic and pharmacodynamiccharacteristics of the particular agent, and its mode and route ofadministration, as well as the age, weight, and health of the recipient;the nature and extent of the disease; the frequency and duration of thetreatment; the type of, if any, concurrent therapy; and the desiredeffect. It is expected that a useful dosage contains between about 0.1to 100 mg of active ingredient per kilogram of body weight. Ordinarily,0.5 to 50 mg, and preferably 1 to 10 mg of active ingredient (e.g., aprogestin) per kilogram of body weight per day, given in divided dosesor in sustained release form, is appropriate. The compound may beadministered to a patient by any appropriate mode, e.g., orally,transmucosally, transdermally, subcutaneously, intramuscularly, orintravenously, as determined by one skilled in the art. Implants whichdeliver a steady low dosage over a long period of time are particularlydesirable. Appropriate types of formulations for each of such deliveryroutes are well known, or can be derived using standard methods.

[0025] Described below is evidence that progesterone can inhibit VSMCproliferation. The experimental procedures and data are meant toillustrate, but not limit, the methods of the invention. Other suitablemodifications and adaptations of the variety of conditions andparameters of the following procedures are within the spirit and scopeof the present invention.

Experimental Data Materials and Methods

[0026] Immunocytochemistry

[0027] Adult female rat aortas were fixed in 4% paraformaldehyde. Theaorta from a premenopausal woman (provided by the Beth Israel Hospital,Boston) was fixed in 10% formalin. The aortas were processed forparaffin embedding in an automated system (Hypercenter XP, ShandonScientific Ltd) and cut at a thickness of 5 μm. Cultured RASMCs andHASMCs (grown on cover slips) were rinsed three times with PBS, fixedwith phosphate-buffered 4% paraformaldehyde for 10 min, and then washedextensively with PBS. Immunocytochemical analysis for PgRs was performedas described (Lee et al., Proc. Natl. Acad. Sci. USA 87, 5163-5167(1990); Yoshizumi et al., J. Clin. Invest. 95, 2275-2280 (1995)).Polyclonal rabbit anti-human PgR antibody [PR (C-20), Santa Cruz,Calif.] was applied at a 150 μg/μl dilution, and goat anti-rabbitsecondary antibody (IgG H+L, Vector Laboratories, Burlingame, Calif.)was applied at a 1:200 dilution. To control for PgR specificity,arterial tissue (FIG. 1b) and cultured arterial smooth muscle cells (notshown) were also incubated with anti-PgR antibody that had beenpreabsorbed with the PgR antigen [40 μg/ml; PR (C-20 P), Santa Cruz,Calif.]. PgR staining was developed by the avidin-biotin horseradishperoxidase (ABC) method, with 3,3′-diaminobenzidine/nickel sulfate usedas chromogen to yield a black reaction product. Tissue wascounter-stained with methyl green to visualize nuclei. Staining forα-actin (a marker for smooth muscle cells) was visualized by animmunophosphatase technique, with alkaline phosphatase used as chromogento yield a pink reaction product.

[0028] Cell culture

[0029] RASMCs were harvested from the thoracic aortas of adult maleSprague-Dawley rats (200-250 g) by enzymatic dissociation as described(Tsai et al., Proc. Natl. Acad. Sci. USA 91, 6369-6373 (1994)). Thecells were grown in DMEM supplemented with 10% FCS, penicillin (100U/ml), streptomycin (100 μg/ml), glutamine (200 nM), and 25 mM Hepes (pH7.4) in a humidified incubator (37° C., 5% CO₂). HASMCs (Clonetics, SanDiego) were grown in M199 medium (GIBCO BRL, Grand Island, N.Y.)supplemented with 20% FCS, penicillin (100 U/ml), streptomycin (100μg/ml), and 25 mM Hepes (pH 7.4). Cells from passages 5-9 were used.

[0030] [³] thymidine Incorporation

[0031] RASMCs and HASMCs were cultured in 24-well plates containing anappropriate growth medium (DMEM plus 10% FCS for RASMCs, and M199 plus20% FCS for HASMCs). After the cells had grown to 70-80% confluence,they were rendered quiescent by incubation for 72 h in DMEM (for RASMCs)or M199 (for HASMCs) containing 0.4% charcoal/dextran-treated FCS (FIGS.2a and 2 d) or 0.4% calf serum (CS) (FIGS. 2b and 2 c). Phenol red-freeDMEM was used in the experiments with estradiol (FIG. 2a). Water-solubleprogesterone (Sigma), estradiol (Sigma), 2-hydroxypropyl-β-cyclodextrin(vehicle used to make progesterone and estradiol water soluble; Sigma),or PBS (control) was added to the cells at various concentrations andthe cells were incubated for an additional 24 h. In some cases (FIG.2b), cells were treated with the progesterone receptor antagonist RU486for 1 h and then incubated with progesterone for another 24 h. Fordetermining the effects of serum concentration on progesterone-induced[³H] thymidine incorporation (FIG. 2c), FCS in various concentrationswas added right after the addition of progesterone. During the last 3 hof the 24-h incubation with progesterone, cells were labeled with[methyl-³H] thymidine (DuPont-NEN, Boston, Mass.) at 1 μCi/ml (1 μCi=37kBq). After incubation, the cells were washed with Dulbecco's PBS, fixedin cold 10% TCA for at least 2 h, and then washed with 95% ethanol.Incorporated [³H] thymidine was extracted in 0.2 N NaOH and measured ina liquid scintillation counter.

[0032] Cell Counting

[0033] RASMCs (2×10⁴) were seeded onto 6-well plates and grown in DMEMsupplemented with 10% FCS. Twenty-four hours later, cells in one platewere released for counting (FIG. 3a, day 0) and the remaining plateswere treated with fresh medium (DMEM supplemented with 1% or 2%charcoal/dextran-treated FCS) and progesterone (3 wells) or vehicle (3wells). Medium and progesterone were changed daily. Cells were countedin a Coulter apparatus at various times after their removal from theplates with trypsin-EDTA.

[0034] Viability Assay

[0035] RASMCs were applied to 24-well plates in growth medium (DMEM plus10% FCS). After the cells had grown to 70-80% confluence, they wererendered quiescent by incubation for 72 h with DMEM containing 0.4% CS.A fresh medium (phenol red-free DMEM supplemented with 2%charcoal/dextran-treated FCS) was applied and a water soluble-form ofprogesterone (Sigma) was added at 0, 5, 50, and 500 nM, and the cellswere incubated for an additional 24 h. Cell viability was determinedwith a modified 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MTT) assay, which is based on the conversion of the tetrazoliumsalt 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2-H-tetrazoliumby the mitochondrial dehydrogenase to a formazan product, as measured atan absorbance of 490 nm. Four samples were analyzed in each experiment.

[0036] RNA Preparation and Northern Analysis

[0037] Total RNA from RASMCs was prepared from cultured RASMCs byguanidinium isothiocyanate extraction and centrifugation through cesiumchloride. Before RNA extraction, cells were made quiescent for 72 h with0.4% CS and then incubated for 24 h with 2% charcoal/dextran-treated FCSand vehicle or various doses of progesterone. RNA samples (20 μg/lane)were electrophoresed on 1.3% formaldehyde-agarose gel, and thentransferred onto nitrocellulose filters. The filters were hybridized to³²p-labeled human cyclin probes as described (Lee et al., J. Biol. Chem.266, 16188-16192 (1991); Lee et al., J. Biol Chem. 269, 12032-12039(1994)). To normalize RNA content of each lane, the filters were washedat 80° C. in 50% formamide solution to remove the cyclin probes, andthen hybridized with oligonucleotide probes for 28S RNA. Hybridizedfilters were washed in 30 mM NaCl, 3 mM sodium citrate, and 0.1% sodiumdodecyl sulfate at 55° C. Radioactive signals were visualized on KodakXAR film or PhosphorImager screens (Molecular Dynamics, Inc. Sunnyvale,Calif.).

Results

[0038] Vascular Smooth Muscle Cells Express the Progesterone Receptor

[0039] The presence of PgRs in the medial layer of the aorta (Ingegno etal., Lab. Invest. 59, 353-356 (1988)) suggests that they are expressedby HASMCs. To confirm that such PgR-positive cells are indeed smoothmuscle cells, we double-stained human and rat tissue, cultured HASMCs,and RASMCs for PgRs and α-actin (a marker of smooth muscle cells).PgR-positive nuclei appeared in the medial layer of an arterial tissuesection from a premenopausal woman (FIG. 1a), and the staining wasblocked in a sequential section treated with anti-PgR antibody that hadbeen preabsorbed with PgR antigen (FIG. 1b). In another sequentialsection (FIG. 1c), PgR-positive nuclei (black) colocalized withα-actin-positive cells (pink). PgR-positive nuclei were also present inthe medial layer of a section of rat arterial tissue (FIG. 1d). Inculture, HASMCs (FIG. 1e) and RASMCs (FIG. 1f) both expressed PgRs intheir nuclei.

[0040] Progesterone Inhibits [³H] thymidine Incorporation in ArterialSmooth Muscle Cells

[0041] Changes in [³H] thymidine incorporation in response toprogesterone were next examined in RASMCs and HASMCs. Progesterone, butnot estradiol or 2-hydroxypropyl-β-cyclodextrin (the vehicle in whichthe progesterone and estradiol were made water soluble), inhibited [³H]thymidine incorporation in RASMCs (FIG. 2a), and this inhibition wasdose-dependent. To confirm the specificity of progesterone's inhibitoryeffect on [³H] thymidine incorporation, we preincubated RASMCs with theprogesterone receptor antagonist RU486. Although RU486 by itself hadlittle effect on [³H] thymidine incorporation, it antagonized theinhibition of [³H] thymidine incorporation induced by progesterone (FIG.2b). The inhibitory effect of progesterone on RASMCs could becounteracted by serum in a dose-dependent manner (FIG. 2c). Aprogesterone-induced, dose-dependent inhibition of [³H] thymidineincorporation also occurred in HASMCs (FIG. 2d).

[0042] Progesterone Inhibits Arterial Smooth Muscle Cell Proliferation

[0043] To confirm further that progesterone inhibits smooth muscle cellproliferation, the effect of progesterone on the growth rate of RASMCswas examined. At FCS concentrations of 1% and 2%, the growth of RASMCstreated with progesterone (500 nM) decreased in comparison with cellstreated with vehicle (500 nM) (FIG. 3a). Cell numbers diverged by thesecond day of culture; and at day 8, the number of progesterone-treatedRASMCs was only 40-50% of that of vehicle-treated cells (FIG. 3a). Thisprogesterone-induced reduction in cell growth was also dose-dependent(FIG. 3b), and was consistent with the inhibitory effect of progesteroneon [³H] thymidine incorporation.

[0044] To confirm that the inhibition of DNA synthesis and cellproliferation by progesterone was not due to cell death caused byprogesterone treatment, a viability assay was conducted. No significantdifference in viability between vehicle-treated and progesterone-treatedRASMCs (at progesterone concentrations of 0, 5, 50, or 500 nM) wasobserved.

[0045] Progesterone Downregulates Expression of Cyclin A and E mRNAs inArterial Smooth Muscle Cells

[0046] Progression of the cell cycle is regulated by the sequentialexpression of cyclins. To determine the effect of progesterone on cyclinexpression, cyclin mRNAs in RASMCs were examined. Twenty-four hoursafter progesterone treatment, the levels of cyclin A and E mRNAs weredownregulated (FIG. 4). In contrast, the levels of cyclins B and D1 mRNAdid not change. These data suggest that progesterone may inhibitarterial smooth muscle cell proliferation by interrupting the cell cycleat the G1/S transition.

Other Embodiments

[0047] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not to limit thescope of the invention, which is defined by the scope of the appendedclaims.

[0048] Other aspects, advantages, and modifications are within the scopeof the following claims.

What is claimed is:
 1. A method of treatment comprising: identifying amammal that is suspected of having, or at a risk of having, a conditioncharacterized by vascular smooth muscle cell proliferation and that isnot being treated with estrogen or an agonist thereof; and administeringto the mammal, in the absence of estrogen or an agonist thereof, anamount of progesterone or an agonist thereof effective to decreasevascular smooth muscle cell proliferation in the mammal.
 2. The methodof claim 1, wherein the mammal is a man or a premenopausal woman.
 3. Themethod of claim 1, wherein the condition is transplant arteriosclerosis.4. The method of claim 1, wherein the condition is atherosclerosis. 5.The method of claim 1, wherein the condition is angioplasty restenosis.6. The method of claim 1, wherein the condition is cardiac vein bypassstenosis.
 7. A method of identifying a compound potentially useful forinhibiting vascular smooth muscle cell proliferation, said methodcomprising: contacting a progesterone receptor with a test compound; anddetermining whether the test compound binds to the receptor, whereinbinding of the test compound to the receptor is an indication that thecompound is potentially useful for inhibiting vascular smooth musclecell proliferation.
 8. The method of claim 7, wherein the progesteronereceptor is in a cell.
 9. The method of claim 8, wherein the cell is amammalian cell.
 10. The method of claim 9, wherein the mammalian cell isa vascular smooth muscle cell.
 11. The method of claim 7, said methodfurther comprising determining whether the test compound activates aprogesterone receptor harbored within a cell, wherein activation of thereceptor by the test compound is a further indication that the testcompound is potentially useful for inhibiting vascular smooth musclecell proliferation.
 12. The method of claim 11, wherein the cell is amammalian vascular smooth muscle cell.
 13. The method of claim 12,wherein said activation is indicated by a decreased level of cyclin A inthe mammalian cell as compared to a control untreated with the testcompound.
 14. The method of claim 12, wherein said activation isindicated by a decreased level of cyclin E in the mammalian cell ascompared to a control untreated with the test compound.
 15. A method ofidentifying a compound potentially useful for inhibiting vascular smoothmuscle cell proliferation, said method comprising: contacting a cellcontaining a progesterone receptor with a test compound; and determiningwhether the test compound activates the receptor, wherein activation ofthe receptor by the test compound is an indication that the compound ispotentially useful for inhibiting vascular smooth muscle cellproliferation.
 16. The method of claim 15, wherein the cell is amammalian cell.
 17. A method of identifying a progesteronereceptor-binding compound potentially useful for inhibiting vascularsmooth muscle cell proliferation, said method comprising: contacting amammalian cell containing a progesterone receptor with a test compoundthat binds the progesterone receptor; and determining whetherproliferation of said mammalian cell is inhibited.
 18. The method ofclaim 17, wherein said mammalian cell is a vascular smooth muscle cell.19. A method of identifying a progesterone receptor-binding compoundpotentially useful for inhibiting vascular smooth muscle cellproliferation, said method comprising: contacting a mammalian cellcontaining a progesterone receptor with a test compound that binds theprogesterone receptor; and measuring a level of cyclin A in the cell,wherein a decrease of said level as compared to an untreated control isan indication that the test compound is potentially useful forinhibiting vascular smooth muscle cell proliferation.
 20. The method ofclaim 19, wherein the mammalian cell is a vascular smooth muscle cell.21. A method of identifying a progesterone receptor-binding compoundpotentially useful for inhibiting vascular smooth muscle cellproliferation, said method comprising: contacting a mammalian cellcontaining a progesterone receptor with a test compound that binds theprogesterone receptor; and measuring a level of cyclin E in the cell,wherein a decrease of said level as compared to an untreated control isan indication that the test compound is potentially useful forinhibiting vascular smooth muscle cell proliferation.
 22. The method ofclaim 21, wherein the mammalian cell is a vascular smooth muscle cell.